1. Field of the Invention
The present invention relates to methods and apparatus for the filtration of micro-organisms from a sample and the culture of the micro-organisms in situ on the filter used in the filtration.
2. Description of the Related Art
As discussed in EP0150775, it is known to capture micro-organisms for culture by the membrane filtration of a sample through a sterile membrane placed on a porous holder. After filtration the membrane may be removed and deposited on to a gelatinous culture medium contained in a Petri dish. The Petri dish may then be incubated at a suitable temperature for the time necessary for the micro-organisms to be able to develop and multiply sufficiently to form colonies visible to the naked eye to permit them to be identified and counted.
Even slightly contaminated samples may be evaluated as the micro-organisms are concentrated on the membrane, and it is possible to filter a significant volume of sample to collect a sufficient number of micro-organisms. However, disadvantages which arise in this method are first that the colonies can run out of nutrients in the local area of the agar medium, which in circumstances where there are large numbers of bacteria on the membrane can result in suppression of growth of individual colonies. Also, care must be exercised in placing the membrane filter on the culture medium to avoid entrapping any air bubbles between the culture medium and the membrane which would prevent contact between the culture medium and a portion of the surface of the membrane, thereby opposing diffusion of the culture medium and inhibiting micro-organism development.
In an alternative procedure, it has been proposed to place the membrane containing the filtered bacteria on to a filter paper wick to conduct nutrient medium to the membrane. However, the amount of medium added to the wick has to be carefully controlled in order that the membrane filter does not become too moist such that confluent growth is observed rather than the growth of individual colonies.
Also , these techniques generally involve the transfer of the membrane from the filtration apparatus to the culturing operation and this additional step has the potential to introduce contamination as the membrane is exposed to the air. It also means that culturing cannot start until the samples have travelled from the sampling site to the laboratory, which take a period of several hours.
As further disclosed in EP-B-0150775, it has been proposed to remedy some of these inconveniences by the use of apparatus consisting of a sterile box with circular elements nested into one another and a removable cover. The box includes an inlet and an outlet disposed on both sides of a holder on which an absorbent pad and a filtration membrane lie, clamped at the periphery of the holder by one of the circular elements. After filtration, a culture medium is introduced through the outlet, that is counter currently to the filtration operation, to saturate the absorbent pad. The box can be placed in an incubator to permit the collected micro-organisms to develop. This avoids the need for any transfer operation after filtration and before culturing and permits samples to be directly taken on the site at which the liquid is collected. However, as stated in EP-B0150775, this method still has serious draw-backs. In particular, the membrane diameter increases when the membrane becomes wetted and since the membrane and the absorbent pad, which are clamped at their periphery to the sterile box, are kept dry prior to use, upon filtration of the sample the wetting of the pad and the membrane may cause the membrane filter to part from the absorbent pad. This will prevent contact between the membrane and the pad saturated with the culture medium and thus disturb the development of the micro-organisms upon incubation.
EP-B0150775 proposes an elaborate solution to this problem which involves sealing a membrane filter across the bottom of a tubular holder such that the holder can be pressurised with air to bulge the membrane outwardly prior to mating it against the surface of a gelled nutrient medium in a cup. This reintroduces the danger of contamination of the system during the manipulation involved. Any micro-organism contamination of the interface between the nutrient medium and the filter membrane is likely to interfere with the growth of the micro-organisms on the opposite face of the membrane filter. Furthermore, the apparatus depicted in EP-B0150775 is unduly elaborate and cumbersome to manufacture and to use.
We have now appreciated that the problem outlined in EP-B0150775 is capable of a substantially simpler solution which avoids reintroducing the draw-backs inherent in the earlier prior art.
Accordingly, the present invention now provides a method for filtering micro-organisms from a sample and culturing the micro-organisms, which method comprises filtering a sample containing micro-organisms through a membrane filter in a filter holder in which holder the membrane filter is supported on an absorbent support, and then supplying culture medium to the micro-organisms on the membrane filter in the filter holder by absorbing the medium into the absorbent support, characterised in that the absorbent support is maintained in an expansible compressed state against the membrane filter.
By virtue of the absorbent support being compressed against the membrane filter, if expansion of the membrane filter takes place any tendency of the membrane filter to expand away from the absorbent support is countered by the absorbent support being able to expand and maintain contact with the membrane filter.
There is therefore no need to remove the membrane filter from the absorbent support after the filtration operation and to mate the surface of the membrane filter against a gel of nutrient medium. Instead, sterile nutrient medium may be applied directly to the compressed absorbent support in the filter holder.
The absorbent support is preferably of reticulated foam. Suitable polymer foams are available made from a wide variety of plastics materials such as polyethers, polyesters, polypropylene, polyvinylchloride and polyurethanes. Preferred polymer foams are of from 50 to 200 ppi (pores per inch) (equivalent to 20 to 80 pores per centimeter) e.g. Approximately 100 ppi (equivalent to 40 pores per centimeter).
Suitably, the absorbent support takes the form of a block of such reticulated foam having a free uncompressed thickness of 0.5 to 3 cm, e.g. about 1 cm and presenting a major face on which the membrane filter may be placed directly or, more preferably, with a layer of a wick material such as filter paper between the membrane filter and the absorbent support.
Preferably, the absorbent support is compressed to a is thickness of from 7/8ths to 1/10th of its free uncompressed thickness. The degree of compression need not be uniform over the whole area of the absorbent support. For instance, if the compression is actually applied at the edges of the absorbent support, the compression in the centre of the absorbent support may well be significantly less.
Preferably, between the membrane filter and the absorbent support of reticulated foam there is an intermediate compression spreading support layer transmitting compression forces applied to the edges of the assembly of the membrane filter, compression spreading layer and absorbent support of reticulated foam so that the centre of the assembly is also under compression. Such a compression spreading layer may conveniently be provided by a sheet of filter paper. Other suitable materials may be employed. They should preferably be flexible and porous. Preferably, such a compression spreading material also serves as a wick to draw culture medium from the absorbent support and to supply it to the membrane filter. It should not obstruct expansion of the absorbent support to match expansion of the membrane filter. It may be chosen to expand as much as the membrane filter when wetted.
A liquid sample to be filtered can be placed in a sample chamber above the membrane filter and can be drawn through the membrane filter by suitable means such as a vacuum pump or syringe. The filtered liquid will be withdrawn through an outlet from the filter holder. Preferably, the culture medium is supplied to the absorbent support for the membrane filter through this same outlet. To facilitate this, the apparatus may be inverted at this stage. Preferably, the culture medium is initially contained in a sealed container in which it can be maintained in a sterile condition and the container of culture medium is connected to the outlet of the filter holder prior to the seal being broken to release the culture medium on to the absorbent support.
The invention includes apparatus for use in filtering and culturing micro-organisms comprising a filter holder defining a flow-path for fluid to be filtered, a filter member in said flow-path comprising a membrane filter supported on an absorbent support, and means compressing said absorbent support against the membrane filter.
Preferably, the filter holder may comprise telescopically interfitting first and second sections, said first section having a floor containing an outlet for filtered fluid and a peripheral wall, said membrane filter and said absorbent support therefore being received in said first section on said floor with said membrane filter further away from said floor, and said second section of the filter holder being received in said first section and compressing said membrane filter and said absorbent support against said floor.
The outlet in the floor of the first section of the filter holder may be bridged by a porous support surface underlying the absorbent support of the membrane filter.
Suitably, the outlet in the floor of the first section of the filter holder is adapted for connection to a syringe by a standard syringe fitting.
The apparatus further includes an assay kit comprising apparatus as described above together with a container of sterile liquid culture medium for introduction on to the absorbent support.
Preferably, said container of culture medium is adapted to mate with the outlet of the first section of the filter holder to allow introduction of the culture medium therethrough on to the absorbent support.
Preferably, means are provided at the outlet of the first section of the filter holder for co-operating with the seal of the container of culture medium to break said seal and release the culture medium after a connection has been established between the container of culture medium and the filter holder.